The present invention relates to methods for preventing graft rejection of transplantated organs, tissues or cells, in particular to such methods which comprise engineering a cell type to express a LAG-3 protein when transplanted in a host. More particularly, the invention relates to the production of a universal gene therapy host cell expressing the LAG-3 protein on its surface.
The lymphocyte activation gene (LAG-3) is a member of the immunoglobulin superfamily, that is selectively transcribed in human activated T (both CD4+ and CD8+) and NK cells (Triebel et al, 1990 ). The sequence data, the compared exon/intron organization, and the chromosomal localization revealed that LAG-3 is closely related to CD4 (Baixeras et al, 1992). The close relationship between LAG-3 and CD4 was further strengthened by the demonstration that both share the same ligand, i.e., MHC class II molecules (Baixeras et al, 1992). However, in contrast to CD4, LAG-3 does not bind the human immunodeficiency virus gp120 (Baixeras et al, 1992). In vivo, LAG-3 expression was neither found in primary lymphoid organs, such as spleen, mucosa-associated lymphoid tissue or normal lymph nodes. However, it was readily detected in inflamed tonsils, or lymph nodes with follicular hyperplasia, supporting the view that even in vivo LAG-3 is expressed following activation (Huard et al, 1994A). Antigen-specific stimulation of T-cell clones in the presence of anti-LAG-3 monoclonal antibody (mAb) led to increased thymidine incorporation, higher expression of activation marker CD25 and enhanced cytokine production (Huard et al, 1994B).
Accordingly, addition of a soluble recombinant form of LAG-3 inhibited antigen-specific T-cell proliferation, suggesting a regulatory role of LAG-3 in CD4+ T-lymphocyte activation (Huard, 1996) and its involvement in extinguishing ongoing immune responses. Recently, it has been shown that LAG-3 also acts as a co-receptor for NK cells and defines different modes of tumor cell killing controlled by the innate immune system (Miyazaki et al., 1996).
The mechanics by which a T cell response to a foreign (allogeneic or xenogeneic) protein or cell or organ is mounted are fairly well understood. Antigen presenting cells (APCs) are attracted to areas of inflammation or damage (that may be induced by surgical transplantation). The repertoire of T cells in the periphery is constantly surveying tissues for evidence of pathogens or the presence of foreign (allo- or xenogeneic) tissue. Once any of these warning signals are recognized, the APCs engulf the protein, digest it and present it to the host""s immune system.
Allogeneic or syngenic tumor cells have been engineered to express viral IL-10 which induces local anergy to the tumors. Such a treatment did not affect the rejection of a non-transduced tumor at a distant site ( Suzuki et al., 1995). IL-10 delivered locally is thought to shift the T cell repertoire reactive to the transplanted cells to a Th2 phenotype that is not cytolytic and may even be protective.
Cells naturally expressing the Fas ligand have been transplanted across allogeneic or exogeneic barriers without immunosuppression. Surveillance of the site of implantation by host T cells results in their killing when contacted by Fas ligand (Bellgrau et al., 1995). Moreover, rejection of pancreatic islet allografts has been prevented by the cotransplantation of syngeneic myoblasts genetically engineered to express the Fas ligand (Lau et al., 1996)
The immune system is well equipped to rapidly identify foreign, diseased or inflamed tissue and rapidly destroys it. This has always been a major barrier to tissue, organ and cell transplantation as well as gene therapy. Major problems are generally associated with chronic immunosuppression, encapsulation or immunoisolation. The unwanted side effects of chronic immunosuppression include increased susceptibility to opportunistic infection and tumor formation.
The desire for long-term acceptance of grafted tissue in the absence of continuous immunosuppression is a long-standing goal in human medicine.
Citation of any document herein is not intended as an admission that such document is pertinent prior art, or considered material to the patentability of any claim of the present application. Any statement as to content or a date of any document is based on the information available to applicant at the time of filing and does not constitute an admission as to the correctness of such a statement.
It has now been found that transplantation of cells that express a LAG-3 protein on their surface results in the protection from graft rejection by the host""s immune system.
The present invention thus provides a genetically engineered cell which may be part of a tissue or organ to be transplanted, comprising DNA encoding a transmembrane LAG-3 protein on its surface, resulting in the protection from graft rejection by the host""s immune system, the DNA being genomic DNA or cDNA. Said DNA can be exogenous or, in a particular embodiment of the invention, the endogenous DNA, whose expression is activated or modified through the targeted insertion of a regulatory sequence and/or an amplifiable gene by homologous recombination. The LAG-3 protein is a protein which is recognized by antibodies directed against LAG-3.
When the cell is part of the tissue or organ to be transplanted, transfection of the LAG-3 DNA can be accomplished directly on the tissue or organ to be transplanted.
In particular, the cell is a universal gene therapy host cell, suitable, for example, for any kind of somatic or xe2x80x9cex vivoxe2x80x9d gene therapy.
In a specific embodiment, the gene therapy host cell further comprises exogenous DNA encoding a therapeutic agent of interest, and the genetically engineered cells are employed as a therapeutic agent. The term xe2x80x9ctherapeuticxe2x80x9d as used herein, includes treatment and/or prophylaxis.
In a further embodiment, the gene encoding a therapeutic agent of interest is present in the genome of the cell and the cell further comprises exogenous DNA encoding a regulatory sequence or an amplifiable gene for activating or modifying the expression of the endogenous gene of interest.
The genetically engineered cell of the invention can, anyway, contain the exogenous LAG-3 DNA only, to be used in a mixture with other gene therapy host cells containing the therapeutic DNA of interest.
The cell of the present invention is preferably selected from myoblasts, fibroblasts, hematopoietic stem cells, embryonic stem cells, foetal liver cells, umbilical vein endothelial cells and CHO cells.
Cells as above, deriving from transgenic animals, are also within the scope of the present invention.
It is a further object of the present invention the use of a transmembrane LAG-3 protein, including muteins and variants thereof, expressed on the surface of the cells, in the manufacture of a medicament to induce protection from graft rejection by a host""s immune system.
Furthermore, the present invention provides the use of a cell comprising DNA encoding a transmembrane LAG-3 protein, expressed on the surface of the cell, in the manufacture of a medicament to induce protection from graft rejection by a host""s immune system.
The use of said cell expressing LAG-3 on its surface, in the manufacture of a medicament to be mixed with cells, tissues or organs to be transplanted, to induce protection from graft rejection by a host""s immune system, is also within the scope of the present invention.